Liposome composition for use in peritoneal dialysis

ABSTRACT

The present invention is directed to a liposome composition for use in the peritoneal dialysis of patients suffering from endogenous or exogenous toxicopathies, wherein the pH within the liposomes differs from the pH in the intraperitoneal cavity and wherein the pH within the liposome results in a liposome-encapsulated charged toxin. The invention also relates to a pharmaceutical composition comprising said liposomes. A further aspect of the present invention relates to a method of treating patients suffering from endogenous or exogenous toxicopathies, preferably selected from drug, metabolite, pesticide, insecticide, toxin, and chemical warfare toxicopathies, more preferably hyperammonemia, comprising the step of administering liposomes of the invention in a therapeutically effective amount into the peritoneal space of a patient in need thereof. Next to human, the present invention is particularly suitable to veterinary aspects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/165,202, filed on Oct. 19, 2018, now pending, which is a continuationof U.S. application Ser. No. 14/420,654, which was granted as U.S. Pat.No. 10,596,114 on Mar. 24, 2020, which is entitled to the benefit under35 U.S.C. § 120 and 365(c) of International Patent Application No.PCT/EP2013/002352, filed Aug. 6, 2013, which claims priority to EuropeanPatent Application No. 12005796.3, filed Aug. 9, 2012, which areincorporated by reference herein in their entireties.

The present invention is directed to a liposome composition for use inthe peritoneal dialysis of patients suffering from endogenous orexogenous toxicopathies, wherein the pH within the liposomes differsfrom the pH in the peritoneal cavity and wherein the pH within theliposome results in a liposome-encapsulated charged toxin. The inventionalso relates to a pharmaceutical composition comprising said liposomes.A further aspect of the present invention relates to a method oftreating patients suffering from endogenous or exogenous toxicopathies,preferably selected from drug, metabolite, pesticide, insecticide,toxin, and chemical warfare toxicopathies, more preferablyhyperammonemia, comprising the step of administering liposomes of theinvention in a therapeutically effective amount into the peritonealspace of a patient in need thereof. Next to human, the present inventionis particularly suitable to veterinary aspects.

BACKGROUND OF THE INVENTION

In order to alter the pharmacokinetic and biodistribution properties ofdrugs, a variety of lipid- and polymer-based particles have beendeveloped and characterized that include liposomes, i.e. small lipidbilayer particles with a diameter in the nanometer to micrometer sizerange, wherein the lipid bilayer surrounds an aqueous environment on theinside. Liposomes are presently used in medicine for drug delivery andin research for drug detoxification.

Drugs can be encapsulated in liposomes by a number of techniques, e.g.by passive methods and pH-gradient techniques. For a review see Fenskeand Cullis, Encapsulation of Drugs within Liposomes by pH-GradientTechniques, Liposome Technology, Volume II, Edited by GregoryGregoriadis, Informa Healthcare September 2006, 27-50. The advantage ofa pH gradient across the lipid bilayer is that once the drug is inside,it can be charged by protonation or deprotonation depending on the drugand the pH, so that its back transfer through the lipophilic membrane tothe outside is hindered. See for example Mayer et al., Biochimica etBiophysica Acta, 857:123-126, 1986 and Madden et al., Chemistry andPhysics of Lipids, 53:37-46, 1990. Therefore, liposomal carrier systemscan significantly buffer the toxicity of drugs by entrapping them intheir lumen. Liposomal drugs are regularly administered intravenously orat the site of intended drug action, e.g. intraperitoneally. Forintraperitoneal liposome drug delivery, reference is made, for example,to Verschraegen et al., J. Cancer Res. Clin. Oncol., 129:549-555, 2003and Parker et al., Cancer Res. 41:1311-1317, 1981. Peritoneal retentionof intraperitoneally administered liposomal{circumflex over ( )}entrapped drugs depends on the composition of the formulation, forexample, the lipid composition of the lipid bilayer, the amount ofcholesterol included, the size of the liposome, the charge of theliposome, and/or the coating of the liposome, e.g. with PEG(polyethylene glycol). Intraperitoneally administered liposomal drugsare subject to blood and lymphatic transport and can be detected inblood, lymph nodes as well as in a number of organs. In particular thesize of liposomes has an influence on peritoneal retention. Forintravenously injected liposomes, a diameter of about 100 nm is oftenconsidered as optimal for prolonged blood circulation, whereasincreasing liposome size produces higher peritoneal retention wheninjected intraperitoneally. Liposomes having a size of about 1000 nm orgreater have the highest peritoneal cavity retention. For furtherinformation on factors influencing the peritoneal retention ofintraperitonally administered liposomes, reference is made to, forexample, Sadzuka et al., Toxicology Letters 1 16:51-59, 2000;Dadashzadeh et al., Journal of Controlled Release, 148:177-186, 2010;Mirahmadhi et al., International Journal of Pharmaceutics, 383:7-13,2010; Hirano and Hunt, Journal of Pharmaceutical Sciences, 74 (9),915-921, 1985.

Moreover, liposomes as well as lipids in emulsion have utility in drugdetoxification.

Jamaty et al., Clinical Toxicology 48:1-27, 2010 review the literatureon the use of intravenous fat emulsions (IFE), i.e. lipid emulsions inthe treatment of acute drug poisoning. Intralipid® is a brand name for aclinically relevant commercial fat emulsion comprising 10, 20 or 30% byweight of purified soy bean oil as well as purified egg phospholipids,glycerin and water for intravenously or parenterally administerednutrition in case of malnourishment. Moreover, it has utility as vehiclefor the anesthetic drugs propofol and etomidate as well as for treatingsevere cardiotoxicity caused by overdose of local anaesthetic drugs suchas bupivacaine to save patients otherwise unresponsive to commonresuscitation methods. For nutritional and antidote therapy, Intralipid®is administered intravenously. Cave and Harvey (Academic EmergencyMedicine, 16:151-156, 2009) reviewed the literature on the use of IFE inantidote therapy. And in an animal model of clomipramineinfusion-treated rabbits, Intralipid® administered intravenously andconcomitantly by peritoneal administration showed an enhancedclomipramine extraction over intravenous administration of Intralipid®alone (see Harvey et al., Academic Emergency Medicine, 16:815-824,2009). The antidote mechanism underlying IFE is that the lipidformulation scavenges and thereby masks the toxic drug by lipidextraction. Of course, this mechanism is only available for drugs withsufficient lipophilicity and depends on the extraction coefficient ofthe drug in the lipid composition.

Like IFE, intravenously administered liposomes are investigated to treatcardiovascular drug intoxication. J.-C. Leroux, Nature Biotechnology,2:679-864, 2007, reviews the use of injectable nanocarriers, inparticular of liposomes for drug detoxification. Bertrand et al., ACSNano, 4 (12), 7552-7558, 2010 demonstrated a detoxification withintravenously administered, transmembrane, pH-gradient liposomes in ratsreceiving intravenous bolus of or perfusion with diltiazem, acardiovascular drug. Unlike the lipid extraction with IFE the liposomalmechanism of action is the liposomal uptake and charging of the drug bythe pH gradient to make the uptake irreversible. Compared to IFE, drugscavenging liposomes are much more efficient in capturing drugs, inparticular calcium channel blockers, see for example, Forster et al.,Biomaterials 33, 3578-3585, 2012.

Hyperammonemia refers to a clinical condition associated with elevatedammonia levels manifested by a variety of symptoms including centralnervous system (CNS) abnormalities. When present in high concentrationammonia is toxic. Endogenous ammonia intoxication can occur when thereis an impaired capacity of the body to excrete nitrogenous waste, asseen with congenital enzymatic deficiencies. A variety of environmentalcauses and medications may also lead to ammonia toxicity. For a reviewof ammonemia reference is made to Auron and Brophy, Pediatr. Nephrol.,27:207-222, 2012; and Clay and Hainline, CHEST, Official journal of theAmerican College of Chest Physicians, (132), 1368-1378, 2007. Usually,hyperammonemia is associated with cerebral edema, decreased cerebralmetabolism and increased cerebral blood flow. Next to therapies thattreat intracranial hypertension, nutritional support to prevent proteincatabolism and stopping nutritional intake of protein, it may benecessary to reduce ammonia levels by actively removing ammonia. Besidesnitrogen elimination through pharmacological manipulation, e.g.administration of sodium phenylacetate and sodium benzoate, to promotethe clearance of ammonia through “alternative” metabolic pathways,peritoneal dialysis, hemodialysis, continuous venovenous hemofiltration,continuous venovenous hemo-diafiltration and continuous arteriovenoushemodiafiltration are effective ways of removing ammonia and have beenhelpful in treating hyperammonemia associated with urea cycle disordersin children and adults. In particular for children with inbornmetabolism errors venovenous haemodialysis and continuous peritonealdialysis are a treatment of choice for the acute management ofhyperammonemia, e.g. see Arbeiter et al., Nephrol. Dial. Transplant.,25:1257-1265, 2010 and Pela et al.; Pediatr. Nephrol., 23:163-168, 2008.

The objective underlying the present invention is the provision of newmedical uses for liposome compositions. Another object is the provisionof new therapies for endogenous and exogenous toxicopathies, inparticular drug, metabolite, pesticide, insecticide, toxin and chemicalwarfare toxicopathies. A further objective of the present invention isthe provision of a new treatment for hyperammonemia.

The above objectives are solved by a liposome composition for use in theperitoneal dialysis of patients suffering from endogenous or exogenoustoxicopathies, wherein the pH within the liposomes differs from the pHin the peritoneal cavity and wherein the pH within the liposome resultsin a liposome-encapsulated charged toxin.

The term “liposome composition for use in the peritoneal dialysis” ismeant to refer to a liposome composition that (i) is suitable forintraperitoneal administration, i.e. it is made of physiologicallyacceptable lipids and further components, (ii) is stable underphysiological conditions, in particular those of the peritoneal cavityand blood, for a time suitable for the uptake and prolonged retention ofdrugs and metabolites, and that (iii) displays a transmembrane transfercapacity.

For practicing the present invention, it is necessary that the pH withinthe liposomes differs from the pH in the peritoneal cavity and that thepH within the liposomes results in a liposome-encapsulated chargedtoxin.

In view of the above, the liposome composition of the present inventionis a physiologically acceptable, stable, transmembrane, pH-gradientliposome composition suitable for peritoneal administration that ispH-adapted for the particular toxin to be charged within the liposome.

The term “peritoneal dialysis” as used herein is meant to be understoodas it is commonly understood by the person skill in the art ofperitoneal dialysis treatment. For practicing the invention, apharmaceutically effective amount of the liposome composition of theinvention is administered into the peritoneal cavity, e.g. by injectionas a single bolus or by continuous infusion or perfusion. The liposomeswithin the cavity and the nearby tissues and organs will take up thetoxin of interest. The pH within the liposome is adapted so that thetoxin is charged upon membrane transfer, i.e. protonated or deprotonatedto result in a positively or negatively charged toxin compound thatcannot transfer back through the hydrophobic liposome bilayer.

The drug-entrapping liposome sequesters the toxin for a prolonged timeperiod and reduces the toxic concentration of the free compound. Theliposome can be left in the peritoneal cavity and body tissues if thetoxic concentration resulting from the eventual biodegradation of theliposome and release of the toxin is not pathological to the patient. Onthe other hand, it is preferred that the liposome composition and/orsize is adapted to prolong peritoneal localization. In that case, it ispreferred that the toxin-loaded liposomes in the abdominal cavity aresuction-extracted from the peritoneal cavity. Intraperitonealadministration and extraction can be performed subsequently and/orsimultaneously.

Physiologically acceptable, transmembrane, pH-gradient liposomecompositions suitable for practicing the present invention can beprepared as abundantly described in the prior art, for example in thedocuments referenced above. The liposome formulation may comprisevesicles of various nature (unilamellar, multilamellar), compositions,sizes, and characteristics, enclosing an aqueous medium of diversecompositions, pH and osmotic strength. In preferred embodiments the mainconstituents of the liposome lipid layer membrane are selected from thegroup consisting of natural or synthetic phospholipids such as thoselisted below:

-   1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC)-   1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC)-   1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC)-   1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC)-   1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC)-   1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE)-   1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE)-   1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE)-   1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE)-   1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (MPPC)-   1-Palmitoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine (PMPC)-   1-Stearoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (SPPC)-   1-Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine (PSPC)-   1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG)-   1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG)-   1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG)-   1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG)-   1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA)-   1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (DPPA)-   1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (DPPS)-   Natural L-α-phosphatidylcholine (from chicken egg, EPC, or from soy,    SPC)

Preferred phospholipids are long saturated phospholipids, e.g. thosehaving alkyl chains of more than 12, preferably more than 14, morepreferably more than 16, most preferably more than 18 carbon atoms.

Preferred liposome compositions for use according to the invention arepreferably those, wherein the liposomes are uni- and/or multilamellar,and comprise

-   (i) 1 to 100, preferably 40 to 70 mol % physiologically acceptable    phospholipids, preferably selected from the group consisting of    DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DSPE, DOPE, MPPC, PMPC,    SPPC, PSPC, DMPG, DPPG, DSPG, DOPG, DMPA, DPPA, DPPS, EPC, and/or    SPC.-   (ii) 1 to 100, preferably 40 to 70 mol % sphingolipids, preferably    sphingomyelin;-   (iii) 1 to 100, preferably 40 to 70 mol % surfactants, preferably    featuring hydrophobic alkyl ethers (e.g. Brij), alkyl esters,    polysorbates, sorbitan esters, and/or alkyl amides;-   (iv) 5 to 100, preferably 50 to 100 mol % amphiphilic polymers    and/or co-polymers, preferably block copolymers comprising at least    one block of a hydrophilic polymer or copolymer such as polyethylene    glycol, and at least one block of a hydrophobic polymer or copolymer    such as poly(lactide), poly(caprolactone), poly(butylene oxide),    poly(styrene oxide), poly(styrene), poly(ethylethylene), or    polydimethylsiloxanes,-   (v) 0 to 60 mol %, preferably 20 to 50 mol % toxin    retention-enhancing compounds, preferably sterol derivatives,    preferably cholesterol,-   (vi) 0 to 30 mol %, preferably 1 to 5 mol % steric stabilizers,    preferably PEGylated compounds, preferably PEGylated lipids, more    preferably DSPE-PEG.

In preferred embodiments liposome-like vesicles are made from polymersand comprise no lipids, for which reason they are formally notconsidered liposomes and are called polymersomes. However, for thepurpose of the present invention polymer-somes are meant to beencompassed by the term liposome as used for defining the invention andthe claims.

Similarly, liposome-like vesicles made from synthetic surfactants andcomprising no lipids are called niosomes. However, for the purpose ofthe present invention nio-somes are meant to be encompassed by the termliposome as used for defining the invention and the claims.

In a preferred embodiment the liposomes for use in the inventioncomprise 10 to 100, more preferably 30 to 80, more preferably 40 to 70,most preferably 50 to 60 mol % of physiologically acceptablephospholipids.

In a preferred embodiment the liposomes for use in the inventioncomprise 10 to 100, more preferably 25 to 75, more preferably 40 to 70,most preferably 50 to 60 mol % of sphingolipids, preferablysphingomyelin.

In a preferred embodiment the liposomes for use in the inventioncomprise 30 to 100, more preferably 40 to 95, most preferably 45 to 60mol % of surfactants.

In a preferred embodiment the liposomes for use in the inventioncomprise 5 to 00, more preferably 30 to 100, more preferably 60 to 100,most preferably 95 to 100 mol % of amphiphilic polymers and/orcopolymers.

In preferred embodiments the concentration of the sterols in theliposome composition varies between 0 and 60, preferably 20 and 50, morepreferably 30 to 45 mol % for enhanced retention of the metabolite ordrug.

In a further preferred embodiment the concentration of the stericstabilizer, preferably PEGylated lipids, in the liposome compositionvaries between 0 and 30, preferably 0.5 and 20, more preferably 1 to 5mol %.

In another preferred embodiment the diameter size of the liposomes islarger than 600, preferably larger than 700, most preferably larger than800 nm, i.e. a diameter size of 600 nm to 10 μm, preferably 700 nm to 10μm, more preferably 800 nm to 5 μm to avoid too rapid drainage from theperitoneal space.

The aqueous solution within the lipid bilayer of the liposome of theinvention is preferably isotonic, preferably has a high bufferingcapacity at low pH (e.g. citrate, sulfate, acetate, benzoate, formate,glycolate, malate buffer) for a high retention of basic compounds and ahigh buffering capacity at high pH (e.g. calcium acetate, bis-trispropane, sulfonates (CAPS, CABS, TABS, CHES), bicine, tricin,ethanolamine buffer) for a high retention of acidic compounds. The pHand buffering compounds have to be adapted to the toxin of interest. Forexample, ammonium sulfate, i.e. a weak base, would not be suitable forscavenging the strong base ammonia (i.e. treatment of hyperammonemia),because it would introduce ammonia into the body, a compound which isaimed at being eliminated. For the sequestration of weak acids, theinternal aqueous solution should be a basic buffer (e.g. calciumacetate).

Preferably, the liposome composition of the invention features a pHwithin the liposome composition of 1 to 6.5, preferably 1.5 to 5, morepreferably 1.5 to 4.

Also preferred is that the liposome composition for use in the inventionfeatures a pH within the liposome composition of 8.5 to 12, preferably 9to 11, more preferably 9 to 10.

In a most preferred embodiment, the liposome composition for use in theinvention is one wherein the liposome bilayer comprises:

-   -   (i) 50 to 60, preferably about 54 mol % DPPC,    -   (ii) 40 to 50, preferably 45 mol % cholesterol (CHOL),    -   and 0.5 to 2, preferably 1 mol %        1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene        glycol)-2000] (DSPE-PEG), and the aqueous solution within the        liposomes comprises 250 mM citrate solution buffered at pH 1.5        to 3, preferably 2,    -   and wherein the diameter of the liposomes is 800 nm or larger,        preferably 900 nm or larger, more preferably 1000 nm or larger.

The above liposome composition is preferred for treating hyperammonemia.Further examples of liposomes suitable for practicing the invention are(in molar ratios): DMPC/CHOL, 2:1; DPPC/CHOL, 2:1; DSPC:CHOL, 2:1;DSPC/CHOL/PEG-2000-DSPE, 2:1:0.2; DSPC/DSPG/CHOL, 60:10:30;DSPC/DSPG/CHOL/PEG2000-DSPE, 55.83:10:27.92:6.25, EPC/CHOL/DESP-PEG,50:45:5, sphingomyelin/DPPC/-CHOL/DSPE-PEG, 25:25:45:5, comprising andinternal buffer of citrate 110 mM pH 4, or 200 mM pH 3, or 250 mM pH 3,or 250 mM pH 2, or 300 mM pH 2.

The terms exogenous and endogenous toxicopathy indicate the involvementof a toxin produced by the body, i.e. endogenous toxin, or a toxinintroduced from externally into the body, i.e. exogenous toxin.

Preferably, the liposome composition of the invention is for treating anexogenous toxicopathy selected from the group consisting of metabolite,drug, pesticide, insecticide, toxin and chemical warfare toxicopathies.Preferred herbicide intoxications are from auxins or atrazine and apreferred pesticide intoxication is from neonicotinoid.

In a preferred embodiment, the present invention is directed to thetreatment of a metabolite toxicopathy. The term “metabolite toxicopathy”as used herein is meant to indicate a toxic concentration of endogenouscompounds leading to pathological conditions, e.g. ammonia,argininosuccinate, uric acid, isovaleric acid, propionic acid. The termdoes not include pathologies based on exogenous metabolites, e.g. drugmetabolites.

Preferred metabolite toxicopathies are selected from the groupconsisting of hyperammonemia, argininosuccinic acidemia, hyperuricemia,isovaleric acidemia and propionic acidemia.

In a further preferred embodiment, the present invention is directed tothe treatment of a drug toxicopathy, preferably selected from the groupconsisting of acidic and basic drugs.

Drugs suitable for intraperitoneal liposome entrapment according to theinvention are preferably selected from the group consisting of

(1) antineoplastics, preferably selected from the group consisting ofmitoxantrone, epirubicin, daunorubicin, doxorubicin, ciprofloxacin,vincristine, vinorelbine, or vinblastine;(2) local anaesthetics, preferably selected from procaine, lidocaine,bupivacaine, chlorpromazine, midazolam or dibucaine;(3) adrenergic antagonists, preferably propranolol, phenylephrine,alprenolol, atenolol, clenbuterol, salbutamol or timolol;(4) antiarrythmetic agents, preferably quinidine;(5) cholinergic agents, preferably pilocarpine or physostigmine;(6) antidepressants, preferably imipramine, nortriptyline,amitriptyline, bupropion, doxepine, or venlafaxine;(7) antihistamines, preferably diphenylhydramine, or chlorphenamine;(8) antimalarial agents, preferably primaquine, quinine, chloroquine,amodiaquine, or pyrimethamine;(9) antiprotozoan agents, preferably quinacrine;(10) analgesics, preferably codeine, acetaminophen, aspirin, fentanyl,methadone, or pethidine;(11) cardiovascular drugs, preferably diltiazem, verapamil, ordipyridamole;(12) anticonvulsants, preferably valproic acid, or phenobarbital;(13) antipsychotic drugs, preferably quetiapine, chlorpromazine orhaloperidol;(14) anti-anxiety drugs, preferably alprazolam, or diazepam;(15) anti-inflammatory drugs, preferably diclofenac, or ibuprofen;(16) erectile dysfunction drugs, preferably sildenafil, or tadalafil;(17) anti-tuberculosis drugs, preferably ethambutol, isoniazid, orpyrazinamide;(18) neurotransmitters, preferably epinephrine, or norepinephrine;(19) psychostimulants, preferably amphetamine, MDMA, methylphenidate,cocaine, or heroin.

Most preferred drugs involved in intoxications that can be treated withliposome compositions for peritoneal dialysis of the present inventionare: Acetylsalicylic acid, Alprazolam, Amitriptyline, Amlodipine,Amphetamine, Atenolol, Atropine, Bupivacaine, Bupropion, Captopril,Chloroquine, Chlorpheniramine, Chlorpromazine, Chlorpropamide,Clenbuterol, Cocaine, Codeine, Diazepam, Diltiazem, Diphenhydramine,Dipyridamole, Disopyramide, Doxepine, Ethambutol, Fentanyl, Fentanyl,Haloperidol, Heroin, Ibuprofen, Imipramine, Isoniazid, Ketoprofen,Lidocaine, Lorazepam, MDMA, Metformin, Methadone, Methadone,Methylphenidate, Nifedipine, Nortriptyline, Pethidine, Phenobarbital,Phenprocoumon, Procainamide, Propranolol, Pyrimethamine, Quetiapine,Quinacrine, Quinidine, Quinine, Ropivacaine, Sildenafil, Valproic acid,Venlafaxine, Verapamil, Warfarin.

The liposome compositions of the invention are preferably used fortreating human patients suffering from endogenous or exogenoustoxicopathies. Also preferred is the use of liposome compositions fortreating mammals and birds, preferably mammals selected from the groupconsisting of swine, cattle, dog, cat, sheep, goat and horse, etc.suffering from endogenous or exogenous toxicopathies.

A further aspect of the present invention relates to a pharmaceuticalcomposition comprising at least one liposome composition of theinvention and optionally one or more pharmaceutically acceptableexcipients. Pharmaceutical compositions of the present inventiontypically comprise a therapeutically effective amount of a liposomecomposition according to the invention and optionally auxiliarysubstances such as pharmaceutically acceptable excipient(s). Saidpharmaceutical compositions are prepared in a manner well known in thepharmaceutical art. A carrier or excipient may be a liquid materialwhich can serve as a vehicle or medium for the active ingredient.Suitable carriers or excipients are well known in the art and include,for example, stabilizers, suspending agents, osmotic agents (such asglucose solutions, gelatin, xylitol, sorbitol, mannitol, glucose polymer(e.g, icodextrin), or amino acids), antimicrobial preservatives,antioxidants, pH-regulating substances (e.g. sodium and potassiumlactate), coloring agents, etc. The pharmaceutical preparation of theinvention must be suitable for intraperitoneal administration and may beadministered to the patient, preferably a human, in the form ofsolutions, suspensions, or the like.

Another aspect of the present invention concerns a method of treatment,i.e. a method of treating patients suffering from endogenous orexogenous toxicopathies, comprising the step of administering a liposomecomposition according to the invention in a therapeutically effectiveamount into the peritoneal space of a patient in need thereof.

Preferably, the exogenous toxicopathy to be treated is selected from thegroup consisting of drug, pesticide, insecticide, toxin and chemicalwarfare toxicopathies. Also preferred is that the metabolite toxicopathyto be treated is selected from the group consisting of hyperammonemia,argininosuccinic acidemia, hyperuricemia, isovaleric acidemia andpropionic acidemia.

Preferably, the patients for being treated according to the inventionare humans.

In an alternative embodiment of the method of the invention the patientssuffering from endogenous or exogenous toxicopathies are selected frommammals and birds, preferably mammals selected from the group consistingof swine, cattle, dog, cat, sheep, goat, and horse, etc.

In effecting treatment of patients suffering from endogenous orexogenous toxicopathies as described above, a liposome composition ofthe present invention can be administered in any form or mode whichmakes the liposomes or liposome-like vesicles, e.g. polymersomes orniosomes, bioavailable in an effective amount within the peritonealcavity. Preferably intraperitoneal liposome administration is by bolusinjection, infusion and/or perfusion. One skilled in the art in thefield of pre-paring intraperitoneal formulations can readily select theproper form and mode of administration depending upon the particularcharacteristics of the toxin to be sequestered and/or removed.

In a preferred embodiment the method of the invention further comprisesthe step of extracting the liposomes from the abdominal cavity eithersubsequently to and/or simultaneously to the administration step.

In the following, the invention will be illustrated with reference tospecific experimental embodiments and figures, none of which areintended to limit the invention beyond the scope of the appended claims.

FIGURES

FIG. 1 illustrates the sequestration of toxic substances (e.g. drugs orammonia, NH₃) in the peritoneal space by transmembrane pH-gradientliposomes (case of a weak base). The unionized compound diffuses fromthe blood capillaries to the peritoneal space, where it gets trapped inan ionized form (NH₄) in the vesicles. Diffusion continues until theliposomes internal buffering capacity is overwhelmed.

FIG. 2 is a graph illustrating the liposomes drainage from theperitoneal space to the blood after intraperitoneal administration. Anon-exchangeable sterol dye (Cholesteryl BODIPY® FL-C12, Invitrogen) wasincorporated (0.05 mol %) in the liposomal membrane (during the lipidfilm production process). After intraperitoneal administration ofliposomes, the dye fluorescence (λ_(ex)=470 nm, λ_(em)=520 nm) wasmeasured in the plasma aliquots and compared to a calibration curve toobtain the liposomal lipid concentration. Larger liposomes remainedlonger (8 h) in the peritoneal space whereas small liposomes were foundin blood at important concentrations after 4 h. Mean±SD (n=3).

FIG. 3. is a graph showing the in vitro ammonia uptake by pH-gradientliposomes in 50% fetal bovine serum at 37° C. Liposomes exhibited arapid and efficient uptake of ammonia. The initial ammonia and liposomeconcentrations were set at 1.7 and 3.8 mM, potentially tolerating amaximal capture capacity of 0.45 μmol ammonia/μmol lipid. Interestingly,the vesicles sequestered more than the total amount of ammonia loaded inthe system (dashed line). The surplus came from the native ammoniapresent in the serum. The liposome diameter was of 840 nm. Mean±SD(n=6).

FIG. 4 is a graph showing the concentration of ammonia (NH₃) inperitoneal dialysate in the absence (closed triangles) and presence(open triangles) of liposomes. The dialysis fluid was injectedintraperitoneally at t=0 h in healthy rats. The injected liposome dosewas 180 mg/kg, and the lipid concentration in the dialysis fluid was of15 mM. The liposome diameter was of 850 nm.

FIG. 5 is a graph showing the concentration of verapamil (VP) inperitoneal dialysate in the absence (closed triangles) and presence(open triangles) of liposomes. VP was administered by oral gavage at t=0h (50 mg/kg,

), followed by the intraperitoneal injection of the dialysis fluid att=1 h with

. The injected liposome dose was 180 mg/kg, and the lipid concentrationin the dialysis fluid was of 15 mM. The liposome diameter was of 850 nm.

EXAMPLES

In the following examples it was demonstrated that an illustrativeliposome composition (Example 1) can be retained for a prolonged timeperiod in the peritoneal space after intraperoneal administrationdepending on the size of the liposomes (Example 2). These liposomesexhibited a rapid and efficient uptake of ammonia in 50% fetal bovineserum (Example 3). Moreover, these liposomes were capable of entrappingand concentrating ammonia (Example 4) and orally administered drugverapamil (Example 5) in the peritoneal space, thus demonstrating theutility of such liposomes for the detoxification of metabolites anddrugs by intraperitoneal administration.

Example 1—Liposome Composition and Preparation

The formulation tested in the following experiments in vivo was composedof DPPC with 45 mol % of CHOL and 5 mol % of DSPE-PEG. The aqueoussolution within the liposomes was a 250 mM sodium citrate solutionbuffered at pH 2. The formulations were prepared by the lipid filmhydration/extrusion method (Hope M, Bally M, Webb G, Cullis PR.Production of large unilamellar vesicles by a rapid extrusion procedure.Characterization of size distribution, trapped volume and ability tomaintain a membrane potential. Biochim Biophys Acta 1985, 55-65).Lipids, CHOL, and eventually the Cholesteryl BODIPY® FL-C12 dye, fromInvitrogen (0.05 mol %) were dissolved in chloroform which wassubsequently removed under continuous nitrogen flow and high vacuumfor >12 h. The lipid film was hydrated with citrate buffer (250 mM, pH2). The large vesicles were obtained by extrusion through 2 stackedmembranes of 5 m. The transmembrane pH-gradient was established bydialysis in normal saline for >12 h (membrane cut-off: 1000 kDa).

Example 2—Liposomes Drainage from the Peritoneal Space to the Bloodafter Intraperitoneal Administration

Sprague-dawley rats (male, 300 g) were lightly anesthetized by isofluraninhalation (2%) and 20 mL of a solution of icodextrin 7.5% containingliposomes (either 250 or 850 nm of diameter) bearing thenon-exchangeable sterol dye (Cholesteryl BODIPY® FL-C12, Invitrogen,0.05 mol %) in their membrane were slowly injected in the peritonealspace through sterile puncture. Then, blood aliquots of 250 μL weresampled through the tail veins at 15 min, 1, 2, 4, 6, 8, 10, 12, 14, 16h after i.p. injection. Plasma was separated from the blood aliquots bycentrifugation (6000 g for 10 min) and the dye fluorescence measured inplasma at λ_(em)=520 nm (λ_(ex)=470 nm).

Example 3—In Vitro Ammonia Uptake b pH-Gradient Liposomes in 50% FetalBovine Serum

Ammonia (NH3) uptake kinetics were monitored in 50% FBS in side-by-sidediffusion cells (PermGear, Hellertown, Pa.) at 37° C. The liposomes usedin this experiment had a diameter of 850 nm and contained 54 mol % DPPC,45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and an internalcitrate solution (250 mM) buffered at pH 2. The donor compartment(liposome-free) was separated from the receiver compartment (containingliposomes) by a polycarbonate membrane with 100 nm pores. TheNH₃-to-lipid molar ratio was set to 0.45 with an initial NH₃concentration of 1.7 mM in both cells to achieve equilibrium. NH₃ uptakeby the vesicles in the receiver compartment was directly related to thereduction of toxin concentration in the donor cell. Aliquots of 100 Lwere sampled from the donor compartment 3, 30 min, 1, 2, 4, 8, and 24 hafter injection of pH-gradient liposomes in the receiver compartment.NH₃ was then quantified by a colorimetric assay (Berthelot MPE, Violetd'aniline. Repert Chim Appl 1859, 1:284).

Example 4—Concentration of Ammonia in Peritoneal Dialysate in theAbsence and Presence of Liposomes

Sprague-Dawley rats (300 g) were lightly anesthetized with isoflurane(2.5%, 0.6 L/min O2), kept on a warming blanket, and 20 mL of a solutionof icodextrin 7.5% with (or without) liposomes (3 mg/mL) was slowlyinfused in the peritoneal space through sterile abdominal puncture witha 22G silicon catheter (Venflon; Becton Dickinson). The liposomes usedin this experiment had a diameter of 850 nm and contained 54 mol % DPPC,45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and an internalcitrate solution (250 mM) buffered at pH 2. Aliquots of peritonealdialysate were sampled 0.5, 1, 1.5, 2, 3, and 4 h after dialysis onset.The ammonia content in peritoneal fluid samples was assayed by acolorimetric assay (Berthelot MPE, Violet d'aniline. Repert Chim AppI1859, 1:284).

Example 5—Concentration of Verapamil in Peritoneal Dialysate in theAbsence and Presence of Liposomes

One hour after administration of verapamil (50 mg/kg, p.o.) toSprague-Dawley rats (300 g), animals were lightly anesthetized withisoflurane (2.5%, 0.6 L/min O2), kept on a warming blanket, and 20 mL ofa solution of icodextrin 7.5% with (or without) liposomes (3 mg/mL) wasslowly infused in the peritoneal space through sterile abdominalpuncture with a 22G silicon catheter (Venflon; Becton Dickinson). Theliposomes used in this experiment had a diameter of 850 nm and contained54 mol % DPPC, 45 mol % of cholesterol, and 1 mol % of DSPE-PEG, and aninternal citrate solution (250 mM) buffered at pH 2. Aliquots ofperitoneal dialysate were sampled 2, 4, 6, 8, 10, and 12 h after theoral gavage of verapamil. The drug content peritoneal fluids was assayedby HPLC, as described in, e.g. Forster et al., Biomaterials 33,3578-3585, 2012).

1.-12. (canceled)
 13. A method for treating a metabolite toxicopathy ina patient in need thereof by peritoneal dialysis, comprising: the stepof administering a therapeutically effective amount of a liposomecomposition into the peritoneal cavity of said patient, wherein the pHwithin the liposomes differs from the pH in the peritoneal cavity,wherein the pH within the liposomes results in liposomes-encapsulatedcharged metabolite, wherein the metabolite toxicopathy is selected fromthe group consisting of hyperammonemia, argininosuccinic acidemia,hyperuricemia, isovaleric acidemia and propionic acidemia.
 14. Themethod according to claim 13, wherein said metabolite toxicopathy ishyperammonemia, isovaleric acidemia or propionic acidemia.
 15. Themethod according to claim 14, wherein said metabolite toxicopathy ishyperammonemia.
 16. The method according to claim 13, wherein theliposome composition comprises liposomes having a diameter size largerthan 600 nm.
 17. The method according to claim 16, wherein the liposomecomposition comprises liposomes having a diameter size of 600 nm to 10m, 700 nm to 10 m, or 800 nm to 5 m.
 18. The method according to claim13, wherein the pH within the liposomes is 1 to 6.5.
 19. The methodaccording to claim 18, wherein the pH within the liposomes is 1.5 to 5.20. The method according to claim 19, wherein the pH within theliposomes is 1.5 to
 4. 21. The method according to claim 13, wherein thepH within the liposomes is 8.5 to
 12. 22. The method according to claim21, wherein the pH within the liposomes is 9 to
 11. 23. The methodaccording to claim 22, wherein the pH within the liposomes is 9 to 10.24. The method according to claim 13, wherein the liposomes in theliposome composition are uni- and/or multilamellar, and comprise atleast one of: (i) 1 to 100 mol % physiologically acceptablephospholipids; (ii) 1 to 100 mol % sphingolipids; (iii) 1 to 100 mol %surfactants; (iv) 5 to 100 mol % amphiphilic polymers and/or copolymers;(v) 0 to 60 mol % toxin retention-enhancing compounds; or (vi) 0 to 30mol % steric stabilizers.
 25. The method according to claim 24, whereinthe physiologically acceptable phospholipids are selected from the groupconsisting of DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DSPE, DOPE,MPPC, PMPC, SPPC, PSPC, DMPG, DPPG, DSPG, DOPG, DMPA, DPPA, DPPS, EPC,and SPC.
 26. The method according to claim 24, wherein the sphingolipidscomprise sphingomyelin.
 27. The method according to claim 24, whereinthe surfactants are selected from the group consisting of hydrophobicalkyl ethers, alkyl esters, polysorbates, spans, and alkyl amides. 28.The method according to claim 24, wherein the amphiphilic polymersand/or copolymers are selected from the group consisting of blockcopolymers comprising at least one block of a hydrophilic polymer orcopolymer, and at least one block of a hydrophobic polymer or copolymer.29. The method according to claim 28, wherein the at least one block ofa hydrophilic polymer or copolymer comprises polyethylene glycol (PEG).30. The method according to claim 24, wherein the toxinretention-enhancing compounds are selected from the group consisting ofcholesterol and sterol derivatives.
 31. The method according to claim24, wherein the steric stabilizers are selected from the groupconsisting of PEGylated compounds, PEGylated lipids, and DSPE-PEG. 32.The method according to claim 24, wherein the liposome compositioncomprises liposomes having a diameter of 800 nm or larger.
 33. Themethod according to claim 32, wherein the liposome composition comprisesliposomes having a diameter size of 900 nm or larger.
 34. The methodaccording to claim 32, wherein the liposome composition comprisesliposomes having a diameter size of 1000 nm or larger.
 35. The methodaccording to claim 13, wherein the bilayer of the liposomes comprises:(i) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), (ii) cholesterol(CHOL), and (iii)1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG), wherein the liposome composition comprisesliposomes having a diameter of 800 nm or larger.
 36. The methodaccording to claim 35, wherein the aqueous solution within the liposomescomprises a citrate solution buffered at pH 1.5 to
 3. 37. The methodaccording to claim 35, wherein the bilayer of the liposomes comprises0.5 to 2 mol % of DSPE-PEG.
 38. The method according to claim 37,wherein the liposome composition comprises liposomes having a diametersize of 900 nm or larger.
 39. The method according to claim 38, whereinthe liposome composition comprises liposomes having a diameter size of1000 nm or larger.
 40. The method according to claim 24, wherein thephysiologically acceptable phospholipid is DPPC, the toxinretention-enhancing compound is cholesterol, and the steric stabilizeris DSPE-PEG.
 41. The method according to claim 13, wherein the bilayerof the liposomes comprises: (i) 1 to 100 mol % of DPPC; (ii) 0 to 60 mol% of cholesterol; and (iii) 0 to 30 mol % of DSPE-PEG.
 42. The methodaccording to claim 41, wherein the bilayer of the liposomes comprises:(i) 10 to 100 mol % of DPPC; (ii) 0 to 60 mol % of cholesterol; and(iii) 0.5 to 2% mol % of DSPE-PEG.
 43. The method according to claim 13,wherein the patient in need thereof is a human.
 44. The method accordingto claim 13, wherein the patient in need thereof is a mammal or a bird.45. The method according to claim 44, wherein the mammal is selectedfrom the group consisting of swine, cattle, dog, cat, sheep, goat andhorse.